Dendritic cells (DCs) are highly specialized antigen-presenting cells (APCs) that act as cellular bridges between innate and adaptive immunity. Recent efforts have begun to clarify the critical role of DCs in maintaining immune tolerance, especially at mucosal surfaces, where substantial interaction occurs between immune cells and commensal microbiota (and microbiota-derived pattern-associated molecular products (PAMPs)). PAMPs trigger both activating and regulatory signals through pattern-recognition receptors (PRRs), like Toll-like receptors (TLRs), on DCs. Specialized mucosal DC subsets may process signals and elaborate immunomodulatory factors in a way that distinguishes them from peripheral DCs that primarily respond to microbes as pathogens. Because of these contextual differences, understanding how mucosal DCs interact with commensal microbiota in a pro-tolerogenic manner has major clinical implications for mitigation of autoimmune and allergic diseases. We have previously shown the TLR signaling factor TRAF6 is a critical activator of peripheral DCs, but multi-organ defects in TRAF6-null mice have until now prevented examination of in situ effects of DC-expressed TRAF6 on immune tolerance. We have now generated DC-specific TRAF6- deficient mice (TRAF6?DC), which we unexpectedly found exhibit: 1) spontaneous fibrotic eosinophilic enteritis, 2) aberrant mucosal Th2-associated immunity, and 3) defective iTreg development. Strikingly, these phenotypes require commensal microbiota. We therefore propose TRAF6?DC mice as a novel platform for study of DCs in mucosal tolerance, and will pursue the following specific aims: 1. Investigate the mechanism(s) by which commensal gut microbiota interact with DCs to maintain immune homeostasis. To confirm links between DC TRAF6, commensals, and immune tolerance in a more robust system (and to allow for targeted manipulation of commensal microbiota), we first propose re-derivation and phenotypic re-analysis of TRAF6?DC mice under germ-free conditions. We will conduct bacterial re-introduction experiments to track both effects on bacterial load and possible links between TRAF6?DC, immune tolerance, and the hygiene hypothesis. Second, because we have observed higher bacterial loads in TRAF6?DC small intestines we will perform microbiotic profiling to investigate whether DC-intrinsic signals affect gut bacterial population dynamics. 2. Characterize the cellular and molecular mechanisms driving loss of tolerance in TRAF6?DC mice. The molecular functions of TRAF6 and the upstream TLR adaptor MyD88 are closely linked. The microbiota-dependence of the TRAF6?DC gut phenotype implies a key role for DC TLR signaling, but we find that MyD88?DC mice, lacking MyD88 specifically in the DC compartment, do not phenocopy TRAF6?DC. Therefore, we will use genetic models to determine whether: 1) microbiota-derived stimuli regulate gut immune homeostasis directly through DC TLRs using a MyD88-independent pathway, and/or 2) the same stimuli utilize MyD88-dependent pathways in non-DCs in order to access TRAF6-dependent functions within DCs.